EP0835939A2 - Fusion proteins with parts of immunoglobulins, their production and use - Google Patents

Abstract

Soluble fusion protein (A) consists of: (i) a human protein (I), or fragment, not belonging to the immunoglobulin (Ig) family; and (ii) different portions of Ig molecules of any subclass.

Description

The invention relates to genetically generated soluble Fusion proteins consisting of non-immunoglobulin families associated human proteins or parts thereof and various Share the constant region of immunoglobulin molecules. The functional properties of both fusion partners surprisingly remain in the fusion protein.

From EP-A 0325 262 and EP-A 0314 317 are corresponding Fusion proteins consisting of different Domains of the CD4 membrane protein of human T cells and known from human IgG1 fractions. Some of these fusion proteins bind to the glycoprotein with the same affinity gp120 of the human immunodeficiency virus like the cell-bound CD4 molecule. The CD4 molecule belongs to the immunoglobulin family and is therefore very great in terms of its tertiary structure constructed similarly to immunoglobulin molecules. this is also valid for the α chain of the T cell antigen receptor, for those Mergers have also been described (Gascoigne et al., Proc. Natl. Acad. Sci. USA, Vol. 84 (1987), 2937-2940). Because of the very similar domain structure, was therefore in In this case, the maintenance of the biological activity of the both fusion partners can be expected in the fusion protein.

According to the invention preferably at the amino terminus constant region of immunoglobulin coupled human Proteins do not belong to the immunoglobulin family and are assigned to the following classes: (i) membrane-bound proteins, the whole or part of their extracellular domain Fusion is introduced. In particular, these are thromboplastin and cytokine and growth factor receptors like that cellular receptors for interleukin-4, interleukin-7, Tumor necrosis factor, GM-CSF, G-CSF, erythropoietin; (ii) non-membrane-soluble proteins that are whole or partially incorporated into the merger. Especially these are proteins of therapeutic interest such as. Erythropoietin and other cytokines and growth factors.

The fusion proteins can be in known pro- and eukaryotic Expression systems are manufactured, but preferably in mammalian cells (e.g. CHO, COS, BHK cells).

The fusion proteins according to the invention are based on of their immunoglobulin content using affinity chromatography easy to clean and have improved pharmacokinetic properties in vivo.

In many cases the Fc part in the fusion protein is for the use in therapy and diagnostics quite advantageous and leads e.g. to improved pharmacokinetic Properties (EP-A 0232 262). Then again the possibility of removal for some applications of the Fc portion desirable after the fusion protein expressed in the advantageous manner described, has been detected and cleaned. Then this is the Case when the Fc portion is for use in therapy and diagnostics prove to be a hindrance, e.g. if that Fusion protein serve as an antigen for immunizations should.

There are various proteases and their use seems conceivable for this purpose. Papain or Pepsin are used, for example, for the production of F (ab) fragments from immunoglobulins used (Immunology, ed. Roitt, I. et al., Gower Medical Publishing, London (1989)), however, they do not split particularly specific. The blood coagulation factor Xa on the other hand recognizes the relatively rare in a protein Tetrapeptide sequence Ile-Glu-Gly-Arg and performs one hydrolytic cleavage of the protein after the arginine residue by cleavage sequences that the described tetrapeptide were first included by Nagai and Thogersen in one Hybrid protein introduced by genetic engineering (Nagai, K. and Thogersen, H.C., Nature, Vol. 309 (1984), 810-812). These authors were able to show that the in E. coli actually expressed proteins specifically from Factor Xa are split. From publications, however no example is known that such proteins can also be found in eukaryotic and especially expressed in animal cells and cleaved from factor Xa after their purification can be. An expression of the invention However, protein in animal cells is preferable because only in such a cell system the secretion of e.g. normally membrane-bound receptors as fusion partners while maintaining its native structure and so that their biological activity can be expected. Secretion into the cell culture supernatant facilitates the subsequent simple purification of the fusion protein.

The invention thus relates to genetically engineered soluble fusion proteins consisting of non-immunoglobulin family belonging to human proteins or parts of it and different proportions of constant regions of heavy or light chains of immunoglobulins different subclasses (IgG, IgM, IgA, IgE). As The constant part of immunoglobulin is preferred heavy chain of human IgG, particularly preferred of human IgG1, the fusion to the hinge area he follows. In a special embodiment, the Fc part by a built-in factor Xa cleavable cleavage sequence can be easily separated.

The invention further relates to methods for genetic engineering Production of these fusion proteins and their Use for diagnostics and therapy.

Finally, the invention is in further examples explained.

Example 1: Thromboplastin Fusion Proteins

Blood clotting is a central process in the human organism. It is regulated accordingly Coagulation cascade, in which a variety of cellular factors and plasma proteins interact. The entirety of this Proteins (and their cofactors) are called clotting factors designated. The end products of the coagulation cascade are thrombin, which induces platelet aggregation, and fibrin, which stabilizes the platelet thrombus. Thrombin catalyzes the formation of fibrin from fibrinogen and will itself formed by limited proteolysis of prothrombin. For this step activated factor X (factor Xa) responsible in the presence of factor Va and Calcium ions bind to platelet membranes and prothrombin splits.

There are two ways to activate factor X, the extrinsic and the intrinsic "pathway". In the intrinsic "pathway" is a series of factors Proteolysis activated to self-active proteases form. Thromboplastin becomes in the extrinsic "pathway" (Tissue Factor) synthesized by injured cells and activates factor X, along with factor VIIa and Calcium ions. It used to be assumed that the Thromboplastin activity limited to this reaction. However, the thromboplastin / VIIa complex intervenes at the level of factor IX also activating in the intrinsic "pathway". So there is a thromboplastin / VIIa complex one of the most important physiological activators of the Blood clotting.

It is therefore conceivable that, apart from its use as a diagnostic agent (see below), also as a component of therapeutic agents for the treatment of congenital or acquired Blood clotting deficits can be used. Examples include chronic hemophilia due to a lack of factors VIII, IX or XI or also acute blood clotting disorders as a result of e.g. Liver or Kidney disease. Even after surgery the use of such a therapeutic would be conceivable.

Thromboplastin is an integral membrane protein that is not belongs to the immunoglobulin family. Thromboplastin cDNA sequences are published by a total of four groups (Fisher et al., Thromb. Res., Vol. 48 (1987), 89-99; Morrisey et al. 1987 Cell 50: 129-135; Scarpati et al., Biochemistry, Vol. 26 (1987), 5234-5238; Spicer et al., Proc. Natl. Acad. Sci. USA, Vol. 84 (1987), 5148-5152) Thromboplastin cDNA includes an open reading frame that encodes a polypeptide of 295 amino acid residues, of which the N-terminal 32 amino acids act as a signal peptide. Mature thromboplastin consists of 263 amino acid residues and has a three-domain structure: i) amino terminals extracellular domain (219 amino acid residues); ii) transmembrane region (23 amino acid residues); iii) cytoplasmic Domain (carboxy terminus; 21 amino acid residues). In the extracellular domain there are three potential sites for N-glycosylation (Asn-X-Thr). Is thromboplastin usually glycosylated, but glycosylation appears not essential for the activity of the protein (Paborsky et al., Biochemistry, Vol. 28 (1989), 8072-8077).

Thromboplastin is used as an additive to plasma samples in coagulation diagnostics needed. Through the one-step prothrombin coagulation time determination (e.g. quick test) Determine the coagulation status of the examined person. The Thromboplastin required for diagnosis is currently being developed obtained from human tissue using the manufacturing process is difficult to standardize, the yield is low lies and considerable amounts of human raw material (Placentas) must be provided. On the other hand to be expected that the genetic engineering of native, membrane-bound thromboplastin caused by complex cleaning procedures that will be problematic. This Problems can arise from the fusion according to the invention Immunoglobulin fractions are bypassed.

The thromboplastin fusion proteins according to the invention are from mammalian cells (e.g. CHO, BHK, COS cells) into Culture medium discharged via affinity chromatography protein A-Sepharose and surprisingly possess high activity in the one-step prothrombin coagulation time determination.

Cloning of thromboplastin cDNA

The was published for cloning the thromboplastin cDNA Sequence of Scarpati et al., Biochemistry, Vol. 26 (1987), 5234-5238. This resulted in two oligonucleotide probe molecules (see Fig. 1). With these two probe molecules became a human placenta cDNA library (Grundmann et al., Proc. Natl. Acad. Sci. USA, Vol. 83 (1986), 8024-8028) searched.

Different length cDNA clones were obtained. One clone, 2b-Apr5, which is used for the further procedure, codes for the same amino acid sequence as that in Scarpati et al. cDNA described. 2 shows the overall sequence of clone 2b-Apr5 with the thromboplastin amino acid sequence derived therefrom. Construction of one for thromboplastin fusion protein

encoding hybrid plasmid pTF1Fc.

The plasmid pCD4E gamma 1 (EP 0 325 262 A2; deposited with ATCC under number 67610) is used to express a Fusion protein from human CD4 receptor and human IgG1. The DNA sequence coding for the extracellular domain of CD4 is from this plasmid with the restriction enzymes HindIII and BamHI removed. With the enzyme HindIII may only partial cleavage to be carried out here only for one of the two contained in pCD4E gamma 1 Cut HindIII sites (position 2198). Then it lies an open vector, in which a eukaryotic Transcription regulation sequence (promoter) from the open HindIII site is followed. The BamHI position is open at the beginning of the coding regions for one Pentapeptide linkers, followed by the hinge and the CH2 and Human IgG1 CH3 domains. The reading frame in the BamHI recognition sequence GGATCC is such that GAT as Aspartic acid is translated. DNA amplification with thermostable DNA polymerase enables a given Change sequence so that at one or both ends any sequences can be added. Two oligonucleotides were synthesized using sequences in the 5'-untranslated region (A: 5 'GATCGATTAAGCTTCGGAACCCGCTCGATCTCGCCGCC 3') or coding region (B: 5 'GCATATCTGGATCCCCGTAGAATATTTCTCTGAATTCCCC 3') the Can hybridize thromboplastin cDNA. Oligonucleotide A is partially homologous to the coding sequence Stranges, oligonucleotide B is partially homologous to non-coding strand; see Fig. 3.

After amplification, there is a DNA fragment before (827 bp), which (based on the coding strand) on 5 'end before the start of the coding sequence HindIII site, at the 3 'end after the codons for the first three amino acid residues of the transmembrane region a BamHI site contains. The reading frame in the BamHI interface is such that after ligation with the BamHI site in pCD4E gamma 1 a gene fusion is achieved with a continuous Reading frame from the initiation codon of the thromboplastin cDNA up to the stop codon of the IgG1 heavy chain. The Desired fragment was obtained and after treatment with HindIII and BamHI in those described above with HindIII (partially) / BamHI cut vector pCD4E gamma 1 ligated. The resulting plasmid was named pTF1Fc (Fig. 4).

Transfection of pTF1Fc in mammalian cells

The fusion protein encoded by the plasmid pTF1Fc is in the hereinafter referred to as pTF1Fc. pTF1Fc became transient in COS cells expressed. For this purpose, COS cells were used with the help of DEAE-dextran transfected with pTF1Fc (EP A 0325 262).

Indirect immunofluorescence tests showed approximately 25% as Proportion of transfected cells. 24 hours after transfection the cells are transferred to serum-free medium. This cell supernatant was harvested after another three days.

Purification of pTF1Fc fusion protein Cell culture supernatants

170 ml of supernatant from transiently transfected COS cells were overnight in a batch process at 4 ° C with 0.8 ml Protein A Sepharose collected in a column, with 10 volumes Wash buffer washed (50mM Tris buffer pH 8.6, 150mM NaCl) and with elution buffer (100mM citric acid: 100mM NaCitrate 93: 7) in 0.5 ml fractions. The first 9 fractions were immediately neutralized with 0.1 ml of 2M Tris buffer pH 8.6 combined and the protein contained by three cycles of concentration / dilution in the Amicon microconcentrator (Centricon 30) in TNE buffer (50mM Tris buffer pH 7.4, 50mM NaCl, 1mM EDTA). The so obtained pTF1Fc is electrophoretically pure in the SDS-PAGE (U.K. Lambli, Nature 227 (1970) 680-685). In absence from In SDS-PAGE, reducing agents behave like a Dimer (approx. 165 KDa).

Biological activity of purified TF1Fc in the Prothrombin clotting time determination

TF1Fc fusion protein is in low concentrations (> 50 ng / ml) in the one-step prothrombin clotting time determination (Vinazzer, H. Coagulation Physiology and Methods in the blood coagulation laboratory (1979), Fisher Verlag, Stuttgart) active. The clotting times achieved are comparable to the clotting times that with thromboplastin that human placenta was isolated.

Example 2: Interleukin-4 Receptor Fusion Proteins

Interleukin-4 (IL-4) is synthesized by T cells and was originally referred to as B cell growth factor because it can stimulate B cell proliferation. It practices one Variety of effects on these cells. In particular is this stimulates the synthesis of molecules of the immunoglobulin subclasses IgG1 and IgE in activated B cells (Coffmann et al., Immunol. Rev., Vol. 102 (1988) 5). About that IL-4 also regulates proliferation and differentiation of T cells and other haematopoietic cells. It thus contributes to the regulation of allergic and others immunological reactions. IL-4 binds with high Affinity for a specific receptor. The cDNA for encoding the human IL-4 receptor was isolated (Idzerda et al., J.Exp.Med. Vol. 171 (1990) 861-873. From the analysis the amino acid sequence derived from the cDNA sequence shows that the IL-4 receptor consists of a total of 825 amino acids exists, the N-terminal 25 amino acids as Signal peptide act. Mature human IL-4 receptor exists from 800 amino acids and, like thromboplastin, has one Three domain structure: i) amino terminal extracellular domain (207 amino acids); ii) transmembrane region (24 amino acids) and iii) cytoplasmic domain (569 amino acids). In the extracellular domain there are six potential sites for N-glycosylation (Asn-X-Thr / Ser). Has IL-4 receptor Homologies to the human Il-6 receptor, to the β subunit of human IL-2 receptor, for mouse erythropoietin receptor and to the rat prolactin receptor (Idzerda et al., op. cit.). It thus, like thromboplastin, is not a member of the immunoglobulin family, but is together with the listed homologous proteins to the new family of hematopoietin receptors expected. Members of this family are 4 Cysteine residues and one located near the transmembrane region conserved sequence (Trp-Ser-X-Trp-Ser) common in the extracellular domain.

Due to the described function of the IL-4 / IL-4 receptor system is a therapeutic use of a recombinant Form of the IL-4 receptor for the suppression of IL-4-mediated Immune reactions (e.g. transplant rejection reaction, Autoimmune diseases, allergic reactions) possible.

Make the amount of substance required for therapy genetic engineering of such molecules is necessary. According to the invention is because of the easy cleaning Affinity chromatography and the improved pharmacokinetic Properties the synthesis of soluble forms of the IL-4 receptor as an immunoglobulin fusion protein in particular advantageous.

The IL-4 receptor fusion proteins are derived from mammalian cells (e.g. CHO, BHK, COS cells) discharged into the culture medium, via Affinity Chromatography on Protein A Sepharose cleaned and surprisingly possess identical functional properties as the extracellular Domain of the intact membrane-bound IL-4 receptor molecule.

Construction of an for IL-4 receptor fusion protein encoding hybrid plasmid pIL-4RFc.

If the plasmid pCD4EGamma1 is cut with XhoI and BamHI, there is an open vector with the open XhoI site is "downstream" from the promoter sequence. The BamHI site is open at the beginning of the coding regions for a pentapeptide linker, followed by the hinge and the Human IgG1 CH2 and CH3 domains. The reading frame in the BamHI recognition sequence GGATCC is such that GAT is translated as aspartic acid. DNA amplification with thermostable DNA polymerase enables a given Change sequence so that at one or both ends any sequences can be added. Two oligonucleotides were synthesized using sequences in the 5'-untranslated region (A: 5 'GATCCAGTACTCGAGAGAGAAGCCGGGCGTGGTGGCTCATGC 3') or coding region (B: 5 'CTATGACATGGATCCTGCTCGAAGGGCTCCCTGTAGGAGTTGTG 3') the IL-4 receptor cDNA cloned in vector pDC302 / T22-8 is present (Idzerda et al., op. cit.), can hybridize. Oligonucleotide A is partially homologous to the sequence of the coding strand, oligonucleotide B is partially homologous to the non-coding strand; see Fig. 5. After Amplification by means of thermostable DNA polymerase is a DNA fragment before (836 bp), based on the coding strand at the 5 'end before the start of the coding Sequence an XhoI site, at the 3 'end before the last codon contains a BamHI site in the extracellular domain. Of the Reading frame in the BamHI interface is such that after ligation with the BamHI site in pCD4E gamma 1 one Gene fusion is achieved with a continuous reading frame from the initiation codon of the IL-4 receptor cDNA to IgG1 heavy chain stop codon. The desired Fragment was obtained after treatment with XhoI and BamHI in those cut with XhoI / BamHI described above Vector pCD4E gamma 1 ligated. The resulting plasmid was given the name pIL4RFc (Fig. 6).

Transfection of pIL4RFc in mammalian cells

The fusion protein encoded by the plasmid pIL4RFc is hereinafter referred to as pIL4RFc. pIL4RFc became transient expressed in COS cells. For this purpose, COS cells were used with the help of DEAE-dextran transfected with pIL4RFc (EP A 0325 262). Indirect immunofluorescence tests showed approximately 25% as Proportion of transfected cells. 24 hours after transfection the cells are transferred to serum-free medium. This cell supernatant was harvested after another three days.

Purification of IL4RFc fusion protein Cell culture supernatants

500 ml of supernatant from transiently transfected COS cells were overnight in a batch process at 4 ° C with 1.6 ml Protein A Sepharose collected in a column, with 10 volumes Wash buffer washed (50mM Tris buffer pH 8.6, 150mM NaCl) and with elution buffer (100mM citric acid: 100mM NaCitrate 93: 7) in 0.5 ml fractions. The first 9 fractions were immediately neutralized with 0.1 ml of 2M Tris buffer pH 8.6 combined and the protein contained by three cycles of concentration / dilution in the Amicon microconcentrator (Centricon 30) in TNE buffer (50mM Tris buffer pH 7.4, 50mM NaCl, 1mM EDTA) transferred. That so IL4RFc obtained is electrophoretically pure in SDS-PAGE (U.K. Lämmli, Nature 227 (1970) 680-685). In absence from In SDS-PAGE, reducing agents behave like a Dimer (approx. 150 KDa).

Biological activity of purified IL4RFc

IL4RFc protein binds ¹²⁵ I-radiolabeled IL-4 with the same affinity (KD = 0.5nM) as membrane-bound, intact IL-4 receptor. It inhibits the proliferation of the IL-4-dependent cell line CTLLHuIL-4RI clone D (Idzerda et al., Loc. Cit.) In concentrations of 10-1000 ng / ml. In addition, it is excellently suited for the development of IL-4 binding tests, since it can be bound via its Fc part to microtiter plates pre-coated with, for example, rabbit anti-human IgG and also binds its ligand in this form with high affinity.

Example 3: Erythropoietin Fusion Proteins

Mature erythropoietin (EPO) is one of 166 amino acids existing glycoprotein, which is essential for development of erythrocytes. It stimulates the maturation and the Terminal differentiation of erythroid progenitor cells. The Human EPO cDNA was cloned (EPA-0267 678) and encodes the 166 amino acids of the mature EPO and one for the secretion essential signal peptide of 22 amino acids. With the help of the cDNA, recombinant functional EPO produced in genetically modified mammalian cells and for the treatment of anemic symptoms of various origins (e.g. acute kidney failure) be used clinically.

According to the invention is because of the easy cleaning and improved pharmacokinetic properties the synthesis of EPO as an immunoglobulin fusion protein in particular advantageous.

Construction of an erythropoietin fusion protein encoding hybrid plasmid pEPOFc.

This construction was carried out analogously to that in Example 2 (Section: "Construction of a for Hybrid plasmid encoding IL-4 receptor fusion protein pIL-4RFc "). Two oligonucleotides were synthesized, the with sequences near the initiation codon (A: 5'GATCGATCTCGAGATGGGGGTGCACGAATGTCCTGCCTGGCTGTGG 3 ') or the stop codon (B: 5 'CTGGAATCGGATCCCCTGTCCTGCAGGCCTCCCCTGTGTACAGC 3') the EPO cDNA cloned in vector pCES (EP A 0267 678) can hybridize. Oligonucleotide A is partial homologous to the sequence of the coding strand, oligonucleotide B is partially homologous to the non-coding strand; compare Fig. 7. After the amplification is done by means of thermostable DNA polymerase a DNA fragment before (598 bp) that based on the coding strand at the 5 'end in front of the initiation codon contains an XhoI site and at the 3 'end the codon for the penultimate C-terminal amino acid residue of EPO (Asp) is present in a BamHI recognition sequence. Of the Reading frame in the BamHI interface is such that after ligation with the BamHI site in pCD4E gamma 1 one Gene fusion is achieved with a continuous reading frame from the initiation codon of the EPO cDNA to the stop codon of the heavy chain of IgG1. The desired fragment was obtained and after treatment with XhoI and BamHI in the above described vector pCD4E cut with XhoI / BamHI gamma 1 ligated. The resulting plasmid was named pEPOFc (Fig. 8).

Claims (21)

Soluble fusion proteins consisting of non- Human proteins or immunoglobulin family Parts of it and different proportions of immunoglobulin molecules all subclasses. Fusion proteins according to claim 1, characterized in that the immunoglobulin portion is the constant Is part of the heavy chain of human IgG. Fusion proteins according to claim 2, characterized in that the immunoglobulin portion is the constant Part of the heavy chain of human IgG1 or a Protein A binding fragment thereof. Fusion proteins according to claim 2 or claim 3, characterized in that the merger to the "Hinge" region takes place. Fusion proteins according to claims 1-4, characterized in that that fused to immunoglobulin Protein is the extracellular part of a membrane protein or parts of it. Fusion proteins according to claims 1-4, characterized in that that fused to immunoglobulin Protein is the extracellular portion of thromboplastin or parts of it. Fusion proteins according to claims 1-4, characterized in that that fused to immunoglobulin Protein the extracellular portion of a cytokine or Growth factor receptor or parts thereof. Fusion protein according to claim 7, characterized in that the protein fused to immunoglobulin extracellular portion of IL-4 receptor or parts of it is. Fusion protein according to claim 7, characterized in that the protein fused to immunoglobulin extracellular portion of IL-7 receptor or parts of it is. Fusion protein according to claim 7, characterized in that the protein fused to immunoglobulin extracellular portion of G-CSF receptor or parts of it is. Fusion protein according to claim 7, characterized in that the protein fused to immunoglobulin extracellular portion of GM-CSF receptor or parts of it is. Fusion protein according to claim 7, characterized in that the protein fused to immunoglobulin extracellular portion of erythropoietin receptor or parts of it. Fusion protein according to claims 1-4, characterized in that that fused to immunoglobulin Protein is a non-membrane-soluble protein or is part of it. Fusion protein according to claim 13, characterized in that the protein fused to immunoglobulin Cytokine or growth factor or part thereof. Fusion protein according to claim 14, characterized in that the protein fused to immunoglobulin Erythropoietin or part of it. Fusion protein according to claim 14, characterized in that the protein fused to immunoglobulin GM-CSF or G-CSF or part of it. Fusion protein according to claim 14, characterized in that the protein fused to immunoglobulin Interleukin IL-1 to IL-8 or part of it. Fusion protein according to one of the preceding claims 1-17, characterized in that an additional Factor Xa cleavage site between the immunoglobulin part and the non-immunoglobulin portion is inserted. Process for the production of fusion proteins according to one of claims 1-18, characterized in that the DNA coding for these constructs in a Mammalian cell expression system introduced and after Expression using the fusion protein formed Affinity chromatography on the immunoglobulin content is cleaned. Use of the fusion proteins according to one of the claims 1-18 for diagnostics.

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